Figures

- Determination of the substrate-docking site of protein tyrosine kinase C-terminal Src kinase

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Fig. 1. Alignment of the amino acid sequences of Csk, Chk, Src, and Hck in the peptide-binding lobe. Residues that are uniquely conserved in the Csk family are highlighted blue (polar or charged) or red (hydrophobic or Gly).

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Fig. 2. Ala scanning mutagenesis to identify the substrate-docking site on Csk. (A) Ala scanning of polar or charged residues that are uniquely conserved in the Csk family. The activity of the Ala mutants toward an artificial and a physiological substrate are determined. (B) Structure of the Csk peptide-binding lobe. Residues that are potentially part of the substrate-docking site, the catalytic loop, and the activation loop are indicated. (C) Ala scanning of residues in or near the identified substrate-docking site.

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Fig. 3. Effect of DM and QM on Csk kinase function. The details and rationale of the mutations are described in the text. (A) Effect of docking site mutations on Csk phosphorylation of kdSrc. (B) Effect of docking site mutations Csk's ability to inactivate Src.

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Fig. 4. Pull-down assay to determine the interaction of Csk and mutants with kdSrc. (A) Purified GST, GST-Csk, GST-DM, and GST-QM. (B and C) kdSrc pull-down assay with various Csk variants. Each of the purified GST or fusion proteins (100 pmol) was incubated with purified kdSrc (200 pmol) and precipitated with glutathione-agarose. The proteins retained by the beads were analyzed by SDS/PAGE and Coomassie blue staining.

Fig. 6. Structures of Csk catalytic domain and the substrate-docking site. (A) Ribbon structure of Csk with identified residues in the substrate-docking site shown in a ball-and-stick model. Several loop structures relevant to peptide substrate binding and catalysis are indicated by arrows. (B) Surface structure of Csk (yellow) and the substrate-docking site (colored by electrostatic potential). The active site cleft is indicated.